M. ulcerans cultures subjected to the 16S rRNA RT/IS2404 qPCR assay.

Table 1 shows 29 M. ulcerans cultures that were available at the Department of Infectious Diseases and Tropical Medicine (DITM) and the Kumasi Centre for Collaborative Research (KCCR) for development and technical validation of the 16S rRNA RT/IS2404 qPCR viability assay and the corresponding test results. Sequence analysis of 16S rRNA genes from the listed strains revealed 100% nucleotide concordance of the corresponding genomic regions amplified by the 16S rRNA RT-qPCR; no SNPs or mutations were detected, suggesting a high selectivity of the assay. Sequencing primers are described in Table 3[11].aM. ulcerans cultures were available from previous studies from Kamerun (n = 21) and Ghana (n = 4) at DITM [8] or were available at KCCR (n = 4) from the present study. All strains were of human origin (BUD patients) and confirmed by conventional IS2404 PCR and sequencing of rpoB- and rpsL-genes that revealed the M. ulcerans Agy99 wild-type sequences (GenBank accession no. CP000325.1) [11], [12].bResults of the 16S rRNA RT-qPCR of mycobacterial RNA extracts.cResults of the IS2404 qPCR of mycobacterial DNA extracts.dResults of the IS2404 qPCR of genomic DNA (gDNA) wipeout controls (see Protocols S2 and S3); a positive result indicates gDNA contamination of RNA extracts following DNAse digestions, and a negative result indicates RNA extracts free of gDNA.